In vivo binding pair pretargeting

ABSTRACT

A method for in-vivo targeting a functional moiety in a patient by administering a targeting moiety coupled to an affinity component, wherein the targeting moiety has affinity for binding sites in a target area, and administering a binding partner to the affinity component coupled to a functional moiety to localize the functional moiety in the target area. Preferably the targeting moiety is an antibody and the functional moiety is a radiometal when performing in vivo imaging or therapy. The affinity component may be a novel methotrexate analog.

This is a continuation of U.S. Ser. No. 08/140,186, filed Nov. 4, 1993now issued as U.S. Pat. No. 5,578,289, which is a National Phaseapplication of PCT/US93/01858, filed Mar. 3, 1993, which is acontinuation-in-part of U.S. Ser. No. 07/846,453, filed Mar. 4, 1992,now abandoned.

BACKGROUND OF THE INVENTION

Currently, a broad spectrum of diagnostic and therapeutic agents is usedfor in vivo diagnosis and treatment of cancer and infectious diseases.Radionuclides, one important group of pharmaceutical agents, have beenshown to be useful for radioimaging and radiotherapy. Radioimagingcompounds include metal chelates of radioisotopes such as ¹¹¹ In, ⁶⁷ Ga,^(99m) Tc, or ⁵⁷ Co, which are used to detect cancer lesions byintravenous administration. Radiotherapeutic agents, such as metalchelates of ⁹⁰ Y, exert their cytotoxic effects by localized celldestruction via ionizing radiation. Radionuclides, however, suffer froma number of limitations. A particular problem is caused by their toxicside effects, which limit the dosage that may be used safely. In certaincases, adverse side effects are so severe that an effective therapeuticdose cannot be safely administered. Therefore, specific targeting ofradionuclides to internal target sites, such as solid tumors, has becomea major focus of current medical research. The objective of radionuclidetargeting is to improve tumor to normal tissue ratios by concentratingthe radioisotope at the target site, while minimizing its uptake innon-target tissues.

Monoclonal antibodies, reactive with human tumor-associated antigens,provide promising agents for the selective delivery of radionuclides.Various methods have been described for the conjugation of radionuclidesto antibodies. In one procedure, the tyrosine residues of the antibodymolecule are labeled with ¹³¹ I. Alternatively, bifunctional chelatingagents are applied to bind radioisotopes to antibodies. The bifunctionalchelating agents contain as one functional group a chelating moietycapable of forming a tight complex with a metal ion, and as a secondfunctional group a chemically reactive moiety, such as an activatedester, a nitro or amine group, through which the compounds can becoupled to the antibody. Since bifunctional chelator molecules have beenshown to increase the stability of isotope antibody conjugates, thelatter labeling procedure has gained favor in clinical trials. Despitesome promising results, the data from these studies demonstrate that theuse of radioisotope antibody conjugates has several limitations. Themost important limitation is the high nonspecific uptake of theconjugates in normal tissues, such as liver, bone marrow, and kidney,leading to serious toxic side effects. As a result, some investigatorshave resorted to local or regional injections of radioisotope antibodyconjugates in the area of known lesions, neglecting delivery to remotemetastatic sites. Others have used antibody fragments as deliveryagents, which have a lower molecular weight and, therefore, maypenetrate deeper into tumors. However, they also exhibit high uptake incertain normal tissues resulting in a low therapeutic index.

A recent approach to overcoming these problems has been the developmentof bifunctional monoclonal antibodies. Such antibodies have a dualspecificity, with one binding site for a disease site, e.g. a tumortarget, and one binding site for a hapten, which can function as acarrier for a variety of diagnostic and therapeutic agents includingradionuclides. The dual specificity allowed the development of a twostep targeting procedure for radionuclides. First, the anti-hapten,anti-tumor bifunctional antibody is administered and, after a period oftime sufficient for the bifunctional antibody to localize at the tumorsite, the radionuclide-derivatized hapten is injected. This approach hasthe advantage that the non-toxic targeting moiety and the toxicradionuclide-derivatized hapten can be given separately. As a result,large quantities of the targeting moiety can be administered without therisk of serious toxic side effects. Furthermore, increased uptake ratiosand faster localization of the radionuclide can be expected, since theradioactivity is attached to the low molecular weight structure of theradionuclide-derivatized hapten capable of fast distribution through thebody tissues and rapid clearance through the kidneys.

The bifunctional antibody approach, however, suffers from the fact thatthe antibody molecule is composed of two monovalent antibody fragmentswith different specificities. The avidity of monovalent antibodyfragments such as Fab' fragments is orders of magnitude lower than thatof bivalent antibody molecules. The efficacy of the two stepbifunctional antibody approach, however, is dependent on high aviditybinding of the bifunctional antibody to the radionuclide-derivatizedhapten and to extracellular or cell surface antigens at the target site.Moreover, to allow for efficient clearance of non-bound bifunctionalantibody from circulation before injection of theradionuclide-derivatized hapten, a period of 4 to 6 days is required.Using monovalent antibody fragments, complete dissociation of boundantibody molecules from the target sites is expected in this period oftime. A recent study of the kinetics of antibody binding tosurface-immobilized antigen demonstrated that the intact antibody, boundto the surface-immobilized antigen, did not dissociate significantlyover a period of almost 3 days, whereas a monovalent Fab' fragmentprepared from the same antibody dissociated from the surface-boundantigen with a half-life of 16 hours (N. Nygren, C. Czerkinsky, M.Stenberg, Dissociation of antibody bound to surface-immobilized antigen.J. Immunol. Meth. 85, 87-95, 1985).

In addition to the limitation of monovalent binding, there are problemswith the current procedures for the production of bifunctionalantibodies. In one method two Fab' fragments of differing specificityare chemically linked to form a F(ab)₂ fragment with dual specificity.The preparation of appropriate antibody fragments requires individualadjustment of the experimental conditions for each monoclonal antibody,the yields are often very low, and the hybrid antibodies usually suffersignificant, irreversible denaturation. Such denaturation can reduceimmunoreactivity and would be expected to result in different metaboliccharacteristics in vivo. Alternatively, fusion of two hybridomas or ahybridoma with immune spleen cells can be undertaken, with appropriatephysical or biochemical selection of hybrid hybridomas. The theoreticalmaximum yield of bifunctional antibody, produced by established hybridhybridomas, will be 50% of the total immunoglobulin synthesized, theremainder being bivalent parent antibodies. However, the actualproduction of bifunctional antibody can be much lower. In a recent studya bispecific monoclonal antibody against methotrexate and a human tumorassociated antigen was prepared to augment the cytotoxicity of amethotrexate-carrier conjugate. (M. V. Pimm, R. A. Robins, M. J.Embleton, E. Jacobs, A. J. Markham, A. Charleston and R. W. Baldwin, Br.J. Cancer, vol. 61, pp. 508-513, 1990). The proportions of the totalimmunoglobulin recovered from the hybrid hybridoma were 60% monospecificantibody from the original hybridoma cells, 27% monospecific antibodyfrom the immune spleen cells, and only 13% bispecific antibody,suggesting a preferential association of homologous heavy chains. Thesedata demonstrate that it will always be necessary when using thehybrid-hybridoma technique to develop strategies for purification of thebifunctional antibody from parent antibodies being produced by thehybridoma. Since the different antibody molecules from one hybridhybridoma share most properties, an efficient removal of themonospecific antibodies would require two affinity purification steps, atime consuming procedure known to cause partial denaturation of thepurified antibodies.

The problems listed in the foregoing are not intended to be exhaustive,but rather to describe many of the factors that tend to limit thepotential clinical value of the described agents. While the two-stepprocedure, developed for bifunctional antibodies, provides someadvantages over other targeting procedures, there exists a need for amore effective means by which the concentration of a radionuclide oranother diagnostic or therapeutic agent may be maintained at in vivotarget sites for a period of time sufficient to achieve desired results.Further, there exists a need for an effective delivery system consistingof components that can be easily synthesized and purified at highyields.

SUMMARY OF THE INVENTION

One general object of the invention is to provide a delivery system fortargeting therapeutic or diagnostic compound to an in vivo target, whichsubstantially overcomes the limitations known in the prior art. A morespecific objective of the invention is to provide methods and componentsfor selectively targeting radionuclides to solid tumor areas.

This invention comprises a system for in vivo localization using anon-toxic targeting moiety coupled to a non-toxic enzyme, which willlocalize to a target site, and an enzyme inhibitor or enzyme substratederivatized with a functional moiety. On administration, the derivatizedenzyme inhibitor or substrate binds to the localized non-toxic enzymecoupled to the targeting moiety, presenting the functional moiety to thetissue at the target site. In the preferred embodiment the targetingmoiety is an antibody or antibody fragment and the functional moietybound to the enzyme inhibitor is a radionuclide. According to theinvention the targeting moiety and enzyme are both non-toxic andminimally or non-immunogenic when coupled, and the derivatized enzymeinhibitor or substrate is also preferably weakly or poorly immunogenicand non-toxic. A further requirement for the enzyme coupled to thetargeting moiety is that it be essentially absent from circulation orpresent in only very low quantities in circulation. With this inventionthere is rapid and specific localization of the targeting moiety coupledto the enzyme, and relatively rapid clearance and specific targeting ofthe functional moiety-derivatized enzyme inhibitor or substrate withextremely little non-specific binding. By these means highly toxic orotherwise undesirable functional moieties can be used in therapy and inimaging. This invention also comprises a novel methotrexate analoguseful for making the functional moiety derivitized enzyme inhibitor anda stabilized dihydrofolate reductase enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunoreactivity of the SPDP modified 16.88 which wasdetermined by measuring binding to the tumor antigen CTAA-16.88 andcomparing to the activity of native 16.88.

FIG. 2 shows the effects of incorporation of a spacer with a terminalsulfhydryl group through amino groups on dihydrofolate reductase usingSPDP. Sulfhydryl incorporation and protein concentration determinationwere performed as described for the antibody.

FIG. 3 shows the activity of rhDHFR following sulfo-LC-SPDP modificationand following reduction with DTT and evaluated to determine the effectsof the treatment on the activity of the enzyme.

FIG. 4 shows the immunoreactivity of two preparations of 16.88-DHFR.

FIG. 5 shows the number of active rhDHFR molecules per IgM molecule andshows the beneficial effects of using the LC-SPDP spacer compared tonormal SPDP in three different conjugate preparations.

FIG. 6 shows the activities of 16.88-DHFR conjugates prepared withLC-SPDP and SPDP alone. Benefits are seen with the LC-SPDP spacer.

FIG. 7 shows the inhibition of rhDHFR by MTX vs. MTX-DHFR, wherein theconcentration of rhDHFR is 10 nM. At 1×10⁻⁸ M and 5×10⁻⁹ M inhibitorconcentration, the inhibitory effects of DTPA-MTX were virtuallyidentical to MTX inhibition, as indicated by the decreased rates ofdihydrofolate reduction.

FIG. 8 shows the results of MTX inhibition of equivalent activities ofnative rhDHFR and 16.88 bound rhDHFR and indicates that MTX binding isproportional to the reductase activities regardless of whether it isfree or in conjugate form. See Example VI.

FIG. 9, an identical experiment as that shown in FIG. 8 performed usingDTPA-MTX confirmed the methotrexate data. See Example VI.

FIG. 10, the synthesis of DTPA-MTX is shown schematically.

FIG. 11 is a TLC radiochromatograph which shows the migration of ¹¹¹In-DTPA-MTX in the silica gel with an R_(f) of 0.5 to 0.7. Free ¹¹¹ Indoes not migrate from the origin in this system.

FIG. 12 is a bar graph showing clearance of ¹¹¹ In-DTPA-MTX and that the¹¹¹ In-DTPA-MTX and the ¹¹¹ In-DTPA clear from the mice at similar ratesindicating the likelihood of rapid urinary excretion of a DTPA-MTX notbound to antibody-DHFR.

FIG. 13 shows the results of ¹¹¹ In-DTPA-MTX binding to antibody-DHFRbound to tumor cells at different concentrations. See Example IX.

FIG. 14 shows the results of ¹¹¹ In-DTPA-MTX binding to antibody-DHFRbound to tumor cells, using different concentrations of ¹¹¹ In-DTPA-MTX.See Example IX.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preferred methods for localizing radionuclides at an internal targetsite in a patient include two, three, four and five step procedures. Thethree, four and five step embodiments are refinements of the basicconcept.

First, a non-toxic targeting moiety coupled to a non-toxic enzyme isadministered parenterally to a patient and allowed to localizeselectively at the target site. Non-localized circulating molecules ofthe targeting moiety-enzyme conjugate are allowed to clear from thecirculatory system. If necessary, this clearing can be accelerated invivo by complex formation or ex vivo by adsorption to a specific matrixusing binding partners, such as antiidiotypic antibodies or antigens,(second step of the three-step procedure). Thereafter, aradionuclide-derivatized enzyme inhibitor or substrate, specific for theenzyme conjugated to the targeting moiety, is given parenterally.Binding of radiolabeled enzyme inhibitor or substrate to the localizedenzyme-derivatized targeting moiety and rapid clearance of unboundradiolabeled enzyme inhibitor results in selective localization of theradionuclide at the target site.

Additional refinements include scavenging of unbound radionuclides usingchelators as an additional step after administering the radionuclideconjugate. An additional step is also the administration of a blockingagent for enzyme inhibitor or substrate binding sites on cells, so theconjugate will only bind to the previously administered enzyme.Combinations of these procedures are contemplated within the invention.

The targeting moiety is typically an antibody reactive with a humantumor associated antigen. Particularly preferred for use in theinvention are bivalent or multivalent human or chimeric monoclonalantibodies that bind with high avidity to tumor associated antigenslocated in an extracellular area (e.g. necrotic area) or on the cellsurface and are not internalized upon binding to a cell surface antigen.The enzyme moiety preferred for use in the invention is of human originor human-like, either by being genetically conserved or by being from agenetically similar species. An important requirement of the inventionis that the enzyme used in the immunoconjugate must be essentiallyabsent or present in only very low quantities in the circulation,extracellular areas, or on the cell surface of target organs to avoidblocking enzyme inhibitor or non-specific binding. In one embodiment,the enzyme is human dihydrofolate reductase, a single chain molecule ofhuman origin that does not occur in extracellular fluids in measurablequantities. The third component of the targeting system is aradionuclide-derivatized enzyme inhibitor capable of binding with highaffinity to the antibody-conjugated enzyme. Preferred for use in theinvention are small molecular weight inhibitors that allow fastdistribution through the body tissues and quick clearance by excretionof unbound inhibitor. The term enzyme inhibitor used in this inventionencompasses molecules that bind to the enzyme and may augment, reduce,or leave unchanged enzymatic activity. Furthermore, the inhibitormolecule should be suitable for derivatization with radionuclides, e.g.,by covalent attachment of a chelator molecule complexed with aradioactive metal, without impairing its affinity for the enzyme. In thepreferred embodiment the radionuclide-derivatized enzyme inhibitor is aconjugate of methotrexate, a potent inhibitor of human dihydrofolatereductase, and diethylenetriamine-pentaacetic (DTPA) acid complexed with¹¹¹ In or ⁹⁰ Y. Using the gamma-carboxyl residue of methotrexate forconjugation to the chelator, the affinity of the inhibitor todihydrofolate reductase is not affected.

Those skilled in the art will recognize that the present invention isnot limited to the targeting of radionuclides. A variety of diagnosticand therapeutic agents other than radionuclides may be attached to theenzyme inhibitor. Furthermore, two or more diagnostic or therapeuticagent residues may be attached to the inhibitor, for example via anoligomeric or polymeric carrier that is modified by one or more agentresidues. Oligomeric or polymeric carriers useful in this regard includenatural and synthetic molecules such as polypeptides andoligosaccharides. Those skilled in the art will further recognize thatthe invention permits the introduction of additional residues to changethe pharmacokinetic properties of the methotrexate-agent conjugates. Forexample, hydrophilic residues, such as sulfate or sulfonate groups, maybe covalently attached to the conjugates to minimize non-specificbinding to non-target proteins in serum or on cell surfaces, and toprevent cellular uptake in non-target tissues.

Another important requirement of the invention is that the systemcomponents must be non-immunogenic or poorly immunogenic. In the case oftreating humans, the targeting moiety, e.g., an antibody, and the enzymeshould be of human origin, humanized, or human-like, either by beinggenetically conserved or by being from a genetically similar species.Alternatively, components having masked immunogenic epitopes and,therefore, of poor immunogenicity may be used. Also, theradionuclide-derivatized enzyme inhibitor must be essentiallynon-immunogenic. The development of human antibodies against foreignproteins has been demonstrated in many studies. Human anti-mouseantibody formation in cancer patients has been reported after singleinjections of murine monoclonal antibodies. Human anti-mouse antibody(HAMA) formation occurs in up to 50% of cancer patients following singleinjections of murine monoclonal antibodies, (T. J. McCallister. S. E.Halpern, R. O. Dillman, D. L. Shawler, FASEB J. 2, 690, 1988), therebylimiting the applicability of these agents to a period of time requiredfor the development of antibodies.

The targeting system described in this invention provides an affinitysystem that eliminates the serious limitations of currently availabletargeting techniques. Most importantly, all system components are highaffinity components. The use of bivalent (e.g. IgG antibodies) ormultivalent (e.g. IgA or IgM antibodies) agents as targeting moietiesresults in efficient natural clearance of non-bound antibody-enzymeconjugates over a period of several days without risk of completedissociation of bound conjugates from the target sites. The use ofenzyme inhibitors and the corresponding enzymes in an affinity systemoffers several advantages. First, some enzyme inhibitors are known tobind with extremely high affinities to the corresponding enzyme. Forexample, the overall binding constant of methotrexate to humandihydrofolate reductase (K_(off) /K_(on) : 2.1×10⁻¹⁰ M) is rarelymatched by the affinity of anti-hapten monoclonal antibodies. Second,enzyme:enzyme inhibitor systems offer the unique possibility of furtherincreasing affinity by constructing multisubstrate analogue inhibitors(A. D. Broom, "Rational Design of Enzyme Inhibitors: MultisubstrateAnalogue Inhibitors," J. Med. Chem. 32, 2-7, 1989). Recently, amultisubstrate adduct inhibitor of a purine biosynthetic enzyme(glycinamide ribonucleotide transformylase) with a picomolardissociation constant has been synthesized (J. Inglese, R. A. Blatchly,S. J. Benkovic, J. Med. Chem. 32, 937-940, 1989). The inhibitor containsderivatives of the two substrates of the biomolecular, enzyme-catalyzedreaction, 10-formyl tetrahydrofolate and glycinamide ribonucleotide. Thebinding affinity of this multisubstrate inhibitor is approximately3-fold higher than the product of the K_(m) values of the twosubstrates, and 10³ -10⁶ times higher than the binding affinity ofeither substrate. In addition to multisubstrate inhibitors, suicide ormechanism-based inhibitors can be used. These inhibitors requireinteraction with the target enzyme in such a way as to initiate thecatalytic process. As the reaction proceeds, a latent functional group,usually an electrophile, is unmasked within the active site. Alkylationor acylation of a suitably disposed active-site nucleophile inactivatesthe enzyme (R. B. Silverman, S. J. Hoffman, J. Med. Res. Rev. 4, 415,1984). The advantage of suicide inhibitors is that upon binding of theinhibitor to the enzyme a covalent linkage between the two molecules isformed. As a result, radionuclide-derivatized inhibitor molecules boundto targeted antibody-enzyme conjugates cannot dissociate.

DETAILED DESCRIPTION OF THE INVENTION

Multi-substrate Analogues of the Inhibitor

Choice of Antibody

Conventional polyclonal antibodies may be applied as carrier moleculeswithin the concept of the invention. However, monoclonal antibodiesoffer multiple advantages. Each monoclonal antibody is specific for oneantigenic determinant. Thus, with monoclonal antibodies the extent ofnon-specific binding to normal tissues and subsequent toxicity to normalnon-target tissues is reduced. In addition, since unlimited amounts ofeach monoclonal antibody can be produced, all individual preparations ofantibody can be controlled to ensure that antigen specificity remainsconstant over the life of the antibody product. Different monoclonalantibodies specific for different epitopes with the same tissuespecifications may be combined. Thus, when using a monoclonal antibodyor a mixture of monoclonal antibodies the efficacy and control of thedelivery system is improved without sacrificing any contributions to theefficacy of the system that may be obtained with conventional polyclonalreagents.

A preferred approach is to use monoclonal or polyclonal antibodies ofthe same species of origin as the animal receiving therapy. It is notrequired that these antibodies be internalized by the target cell. Forthe most part, with the exception of veterinary applications, the use ofhuman, humanized or chimeric antibodies that are primarily human intheir construction, is most desirable. Many human monoclonal antibodieshave been described. Also, approaches to humanizing antibodies developedfrom lymphoid cells of non-human species and methods using variableregion genes from non-human antibodies genetically coupled to humanantibody constant region genes have been described. The advantages ofthe homologous and genetically engineered antibodies are several. Unlikeheterologous, e.g., murine or rat antibodies, the immune response to thehomologous antibody is minimal. At most, a weak response to idiotypicdeterminants of the human antibody occurs and then only after multiplecycles of administration. In our clinical studies with human monoclonalantibodies we have not detected any induction of an immune response toany region of the antibody, idiotypic, allotypic or framework, evenafter repeated doses of up to 200 mg/week. This advantage allows use ofintact whole immunoglobulin rather than more rapidly metabolizedantibody fragments, allows high doses of intact whole immunoglobulin tobe administered and allows the use of multiple cycles of antibodyadministration. In addition antibodies raised in homologous species haveadditional advantages, as they recognize subtle antigenic differencesnot recognized by heterologous antibodies or even genetically engineeredhuman antibodies.

Antibody may be directed against any target, e.g., tumor, tissue,bacterial, fungal, viral, parasitic, mycoplasmal, histocompatibility ordifferentiation antigens or receptors. Antibody may be from any class,IgG, IgA, IgE or IgM, and a combination of antibodies reactive todifferent antigenic determinants may be used.

The targeting moiety need not be restricted to antibody but may be anysubstance that meets the basic requirements for a targeting moiety inthis invention, as long as there is an affinity for the target tissue.Thus agents that bind specifically to certain tissue receptors such ashormones, lymphokines or certain classes of infectious agents may beused.

Construction of the Antibody-Enzyme Complex

Preparation of the immunoconjugate for our targeting system requiresattachment of an enzymatic or affinity component (AC) to an antibody andforming a stable complex without compromising the activity of eithercomponent. Our strategy involves incorporation of a protected sulfhydrylonto the AC using the heterobifunctional crosslinker SPDP(n-succinimidyl-3-(2-pyridyldithio) propionate and then deprotecting thesulfhydryl for formation of a disulfide bond with another sulfhydryl onthe antibody. Instead of destabilizing the antibody with reducing agentsto generate free sulfhydryls, new sulfhydryls will also be incorporatedonto the antibody using SPDP. In the protected form, the SPDP generatedsulfhydryls on the antibody react with the free sulfhydryls incorporatedonto the AC forming the required disulfide bonds. By optimizing reactionconditions, the degree of SPDP modification of each component can becontrolled, thus allowing maximum incorporation of the AC onto theantibody while maintaining maximum activity of each component. SPDPreacts with primary amines and the incorporated sulfhydryl is protectedby 2-pyridylthione.

If SPDP should affect the activities of either the antibody or the AC,there are a number of additional crosslinkers such as 2-iminothiolane orN-succinimidyl S-acetylthioacetate (SATA), available for formingdisulfide bonds. 2-iminothiolane reacts with primary amines, instantlyincorporating an unprotected sulfhydryl onto the protein. SATA alsoreacts with primary amines, but incorporates a protected sulfhydryl,which is later deacetaylated using hydroxylamine to produce a freesulfhydryl. In each case, the incorporated sulfhydryl is free to reactwith other sulfhydryls or protected sulfhydryl, like SPDP, forming therequired disulfide bond.

Other crosslinkers are available that can be used in differentstrategies for crosslinking our immunoconjugate components.TPCH(S-(2-thiopyridyl)-L-cysteine hydrazide and TPMPH((S-(2-thiopyridyl)mercaptopropionohydrazide) react at the carbohydratemoieties of glycoproteins that have been previously oxidized by mildperiodate treatment, thus forming a hydrazone bond between the hydrazideportion of the crosslinker and the periodate generated aldehydes. Theplacement of this crosslinker on the antibody is beneficial since themodification is site-specific and will not interfere with the antigenbinding site of the antibody. TPCH and TPMPH introduce a 2-pyridylthioneprotected sulfhydryl group onto the antibody, which can be deprotectedwith DTT and then subsequently used for conjugation, such as formingdisulfide bonds between components. If disulfide bonding is foundunsuitable for producing stable conjugates, other crosslinkers may beused that incorporate more stable bonds between components. Theheterobifunctional crosslinkers GMBS(N-gama-malimidobu-tyryloxy)succinimide) and SMCC (succinimidyl4-(N-maleimido-methyl)cyclohexane) react with primary amines, thusintroducing a maleimide group onto the component. This maleimide groupcan subsequently react with sulfhydryls on the other component, whichcan be introduced by previously mentioned crosslinkers, thus forming astable thioether bond between the components. If steric hindrancebetween components interferes with either component's activity,crosslinkers can be used which introduce long spacer arms betweencomponents and include derivatives of some of the previously mentionedcrosslinkers (i.e., SPDP). Thus there is an abundance of suitablecrosslinkers, which could be used; each of which should be selecteddepending on the effects it has on optimal immunoconjugate production.

For our preferred embodiment, we have chosen the recombinant humanenzyme dihydrofolate reductase (rhDHFR) as our affinity component andthe anti-tumor IgM human monoclonal antibody 16.88 as the targetingcomponent. Both components are modified with the SPDP derivativeSulfo-LC-SPDP by formation of a disulfide bond between the components.Sulfo-LC-SPDP is identical in its amino reactivity as SPDP but obtains asulfo group on the succinimidyl group, conferring water solubility onthe crosslinker, thus avoiding the use of organic solvents, which mayhave detrimental effects on the activities of both components. Alsoincluded on sulfo-LC-SPDP is a 5-carbon spacer, which reduces sterichinderance between the components. Four most preferred embodiment wefirst stabilize rhDHFR by covalent conjugation with ANPAP-NADP, asillustrated in Example X.

CHOICE OF ENZYME INHIBITOR MOLECULES

Several considerations are important for the choice of enzyme inhibitorssuitable for use in the present invention. High affinity binding of theinhibitor to the corresponding enzyme is the most important requirement.The overall binding constant (K_(off) /K_(on)) should be in the lownanomolar to picomolar range to guarantee tight binding ofradionuclide-derivatized enzyme inhibitor molecules to targetedantibody-enzyme conjugates. Methotrexate represents one example of suchan inhibitor. Methotrexate binds to human dihydrofolate reductase withan overall binding constant (K_(off) /K_(on)) of 2.1×10⁻¹⁰ M andcompetitively inhibits the enzyme with a K_(i) value of 3.4×10⁻¹² M (M.R. Appleman, N. Prendergast, T. J. Delcamp, J. H Freisheim, R. L.Blakley, "Kinetics of the Formation and Isomerization of MethotrexateComplexes of Recombinant Human Dihydrofolate Reductase", J. Biol. Chem.263, 10304-10313m 1988).

One approach to increasing the affinity of enzyme inhibitors is theconstruction of multisubstrate adduct inhibitors. In principle, suchinhibitors can be designed for any enzyme that binds two or moresubstrates simultaneously (cofactors are considered to be substrates inthis context). This includes, but is not limited to, methyl-, formyl-and acetyl-transferases, dehydrogenases, hydroxylases, kinases, andvarious other enzymes such as dihydropteroate synthase, ATP:L-methionineS-adenosyl transferase, and spermidine synthase. For example,multisubstrate adduct inhibitors for enzymes catalyzing bimolecularreactions can be synthesized by covalent conjugation of both substrates.As demonstrated in several studies, some of these substrate conjugatespossess the binding stabilization of both individual substrates, inaddition to the entropic advantage of reduced molecularity (see, forexample, J. Inglese, R. A. Blatchly, S. J. Benkovic., "A MultisubstrateAdduct Inhibitor of a Purine Biosynthetic Enzyme with a PicomolarDissociation Constant", J. Med. Chem. 32, 937-940, 1989). Typically, thebinding affinity of potent multisubstrate adduct inhibitors is 10³ -10⁶times the binding affinity of either substrate. Another approach toincreasing the affinity of the inhibitor-enzyme interaction is tocombine a multisubstrate adduct inhibitor with an enzyme complexconsisting of two or more copies of the enzyme binding site insufficiently close position to allow the simultaneous binding of theinhibitors coupled together.

Alternatively, suicide or mechanism-based irreversible enzyme inhibitorsmay be used. These inhibitors require catalytic conversion by the targetenzyme. The inhibitor itself is chemically unreactive, but the productof the enzymic conversion is a highly reactive molecule. This productimmediately reacts with an active-site moiety, resulting in covalentattachment of the inhibitor to the enzyme and, thereby, in irreversibleinactivation of the enzyme. Due to this mechanism, the efficacy of theseinhibitor molecules is determined, not only by their binding affinity,but also by their capability to serve as a substrate for the targetenzyme. Enzymes that function by covalent catalysis, especiallypyridoxal phosphate and flavin-linked enzymes, are preferred but not theonly targets for mechanism-based irreversible inhibitors. Examples ofsuch inhibitors are beta, gamma-unsaturated amino acids used for theirreversible inhibition of pyridoxal-linked aspartate aminotransferase,gamma-cysthathionase, and tryptophan synthetase. Other examples include2-chloroallylamine and cis-3-chloroallylamine, irreversible inhibitorsof nonflavin-linked monoamine oxidase and flavin-linked monoamineoxidase, respectively (R. R. Rando. Mechanism-based irreversible enzymeinhibitors. Meth.Enzymol. 46, 28-41, 1977).

Further important considerations for the choice of suitable enzymeinhibitors include a) minimal reactivity with normal tissues, b) lowmolecular weight, c) solubility in aqueous solutions, and d) thefeasibility of chemical conjugation of the inhibitor to effectormolecules without impairment of the binding affinity. Preferred for usein the invention are water-soluble, small molecular weight inhibitorsthat are capable of fast distribution through the body tissues and thatcan be cleared rapidly by the kidneys. In order to prevent thedevelopment of antibodies against radionuclide-derivatized enzymeinhibitor molecules, inhibitors with molecular weights less thanapproximately 5,000 daltons are preferred. In one embodiment of theinvention, methotrexate (L-4-amino-N¹⁰ -methylpteroyl-glutamic acid), awater-soluble compound with a molecular weight of 508.5 daltons, is usedas inhibitor of human dihydrofolate reductase. The gamma-carboxyl groupof the glutamate moiety of this inhibitor can be derivatized withoutimpairing its binding to the enzyme.

Although small molecular weight inhibitors are preferred, enzymeinhibitors with molecular weights larger than 5,000 daltons are alsoincluded in this invention. For example human placental ribonucleaseinhibitor (PRI) is a 50 Kd protein that forms tight complexes with bothsecretory and intracellular ribonucleases (P. Blackburne, S. Moore. In:The Enzymes (P. D. Boyer, ed.) vol. 15, pp. 317-433, Academic Press, NewYork, 1982). As a protein with a molecular weight of 50,000 daltons PRIdoes not meet the desired properties of preferred inhibitors with regardto fast distribution through body tissues and rapid clearance by thekidneys. However, PRI is of human origin and competitively inhibitsRNase A with an extremely low K_(i) value of 4×10⁻¹⁴ M, approaching theaffinity of avidin for biotin. Moreover, PRI binds to human angiogenin,a blood vessel-inducing protein with 35% sequence homology to pancreaticRNase, with an even lower K_(i) value of 7×10⁻¹⁶ M (F. S. Lee, R.Shapiro, B. T. Vallee. Tight-binding inhibition of angiogenin andribonuclease A by placental ribonuclease inhibitor. Biochemistry 28,225-230, 1989).

Choice of Effector Molecules

Effector molecules used in the practice of the present invention arepharmacologically active agents, such as radionuclides, drugs, hormones,and anti-metabolites. They are selected according to the purpose of theintended application, such as whether for imaging or killing tumorcells. Furthermore, the selection involves the consideration ofproperties such as water solubility and the ease of covalent attachmentto enzyme inhibitors without loss of activity.

One important class of therapeutic and diagnostic agents useful in theinvention is chelated metals, including chelated radionuclides usefulfor tumor therapy, such as ¹⁸⁶ Re, ⁹⁰ Y or ²¹² Bi, radionuclides usefulfor radioimaging, such as ^(99m) Tc or ¹¹¹ In, chelated paramagneticions useful for magnetic resonance imaging, such as Gd or Mn, andradio-sensitizing chelated metals, such as chelated iron or ruthenium.Effector molecules may also include, for example, anti-tumor agents,such as DNA alkylating or cross-linking agents, toxins, andanti-microbial agents, such as polyene antibiotics (exemplified byamphotericin B). Finally, a combination of compounds may be used. Thislist of examples is in no way intended to be exhaustive nor meant tolimit the scope of this invention. Many other effector molecules may besuitable for the purposes of the present invention. An advantage of thepre-targeting concept with therapeutic radionuclides is that longerlived isotopes may have a therapeutic advantage. In the future,radionuclides previously considered too long lived forradioimmunotherapy may be preferred (e.g., ²²⁵ Ac, ³² P).

Linkage of Enzyme Inhibitor to Effector Molecule

The methods by which enzyme inhibitors and diagnostic or therapeuticagents may be derivatized and covalently coupled are numerous and wellknown in the art. For example, enzyme inhibitors containing nucleophilicmoieties such as primary amine, a thiol, or a hydroxyl group may bereacted with effector molecules that contain electrophilic moieties orhave been derivatized with such a moiety. Examples of electrophilicmoieties include alkyl halides, alkyl sulfonates, active esters such asN-hydroxysuccinimide esters, aldehydes, ketones, and other electrophilicmoieties such as isothiocyano, maleimido, or carboxylic acid chlorideresidues. Vice versa, effector molecules containing a nucleophilicmoiety can be reacted with an electrophilic moiety on the enzymeinhibitor molecule. Thus, any of a wide range of functional groups onboth the enzyme inhibitor and the effector molecule may be utilized forconjugation, provided these groups are complementary. Alternatively,effector molecules may be coupled to enzyme inhibitors using hetero- orhomobifunctional cross-linking reagents. Suitable reactions would bewell known to one skilled in the art based on the nature of the reactivegroups that are available or have been introduced to both molecules andinformation about the active site requirements of the inhibitor and theeffector molecule.

Preferred for the coupling of radionuclides to enzyme inhibitors arechelating agents capable of forming a tight metal complex with a varietyof pharmaceutically useful metals. Typically, the chelate moiety iscoupled to the enzyme inhibitor by reaction with a nucleophilic moiety,such as a primary amino group, or with an electrophilic moiety, such asan active ester.

EXAMPLE 1

Incorporation of a Spacer with a Terminal Sulfhydryl Group Through AminoGroups on the Antibody Using SPDP

SPDP modified 16.88 was prepared by reacting a 15 molar excess ofsulfo-LC-SPDP with the antibody in 0.1M phosphate, 0.1M NaCl, pH 7.2 for30 min. at room temperature with intermittent mixing. A typical reactioncontained 5 mg of IgM antibody (6.7 nmoles) and 53 μg of sulfo-LC-SPDP(100 nmoles) in a volume of 2 mL. After derivitization, the SPDPmodified antibody was purified on a Sephadex G-25 column equilibrated in0.1M phosphate, 0.1M NaCl pH 7.2, subsequently concentrated on aCentricon-30, and stored at 4° C. at no less than 2 mg/mL. SPDPincorporation was determined by adding dithiothreitol (DTT) to finalconcentration of 10 mM to an aliquot of the SPDP modified antibody andmonitoring the release of 2-pyridylthione at 343 nm. The release of 1mole of 2-pyridylthione is equivalent to the incorporation of 1 mole ofsulfhydryl and can be quantitated with an extinction coefficient of8,080 M⁻¹ cm⁻¹. Protein concentration was determined using the BCAprotein assay and the degree of sulfhydryl incorporation determined.Immunoreactivity of the SPDP modified 16.88 was determined by measuringbinding to the tumor antigen CTAA-16.88 and comparing to the activity ofnative 16.88 (FIG. 1).

EXAMPLE II

Incorporation of a Spacer with a Terminal Sulfhydryl Group Through AminoGroups on Dihydrofolate Reductase Using SPDP

SPDP modified recombinant human dihydrofolate reductase (rhDHFR) wasprepared by reacting a 10 molar excess of sulfo-LC-SPDP with rhDHFR in0.1M phosphate, pH 7.5 for 30 minutes at room temperature withintermittent mixing. A typical preparation contained 0.5 mg of rhDHFR(24 nmoles) and 126 μg of sulfo-LC-SPDP (240 nmoles) in a volume of 2mL. After derivatization, the SPDP modified rhDHFR was purified on aSephadex G-25 column equilibrated in 0.1M phosphate, 0.1M NaCl, pH 7.2and concentrated on a Centricon-3. Sulfhydryl incorporation (FIG. 2) andprotein concentration determination were performed as described for theantibody. Since rhDHFR contains no disulfide bonds, the SPDP'sincorporated onto the enzyme could be deprotected with dithiothreitol(DTT) without detrimental effects to the enzyme. To do this, SPDP-rhDHFRwas treated with DTT at a final concentration of 10 mM for 20 min. atroom temperature in 0.1M phosphate, 0.1M NaCl 1 mM EDTA pH 7.2, purifiedon a Sephadex G-25 column equilibrated in degassed 0.1M phosphate, 0.1MNaCl, 1 mM EDTA, pH 7.2., and then concentrated on a Centricon-3. Afterdetermining the protein concentration by absorbance at 280 nm, thederivatized rhDHFR was immediately used to prepare the finalimmunoconjugate. The activity of rhDHFR following sulfo-LC-SPDPmodification and following reduction with DTT was evaluated to determinethe effects of the treatment on the activity of the enzyme (FIG. 3).

EXAMPLE III

Formation of the Antibody-Enzyme Complex

The immunoconjugate was prepared by adding a 10-15 molar excess ofderivatized rhDHFR to 16.88-SPDP. The reaction was performed in a volumeof 2.5 mL of 0.1M phosphate, 0.1M NaCl, 1 mM EDTA, pH 7.2 at 4° C. for3-4 days under N₂. One to two mg of 16.88-SPDP (1.3-2.6 nmoles) was usedin a typical reaction and the amount or derivatized rhDHFR useddetermined by the antibody quantity used. After incubation at 4° C., themixture was concentrated to 1 mL or less and the immunoconjugatepurified on a Fractogel 55S column equilibrated in 0.1M phosphate, 0.1M,NaCl, pH 7.2. The immunoconjugate was concentrated on a Centriprep-30membrane and stored at 4° C. The immunoreactivity and proteinconcentration were determined as described earlier. FIG. 4 shows theimmunoreactivity of two preparations of 16.88-DHFR. FIGS. 5 and 6 showthe beneficial effects of using the LC-SPDP spacer compared to normalSPDP in three different conjugate preparations. In all cases, the numberof active DHFR's on the IgM was improved by using LC-SPDP.

EXAMPLE IV

Assay of Dihydrofolate Reductase Activity

Dihydrofolate reductase concentrations of 10⁻⁷ -10⁻⁹ M are easilyassayed by monitoring the time dependent decrease in A₃₄₀ caused by thereduction of dihydrofolate and the oxidation of the cofactornicotinadenine dinucleotide phosphate (NADPH). The assay is performed at22° C. (room temperature) in 50 mM Tris, pH 7.5 and 60 μM NADPH andinitiated by adding dihydrofolate to 50 μM. One enzyme unit isequivalent to the amount of enzyme required to reduce 1 μmole ofdihydrofolate to 1 μmole tetrahydrofolate in 1 min. at 22° C. and can bequantitiated using an extinction coefficient of 12,300 M⁻¹ cm⁻¹.

The inhibition rate of DHFR by MTX and its derivatives is determined bythe decrease in the conversion of dihydrofolate to tetrahydrofolate. Theassay conditions are identical to the assay conditions above with onlythe addition of methotrexate or its derivatives at MTX!≦ DHFR!.Derivatives of methotrexate are evaluated by comparing inhibition ratesto the inhibition rate of MTX at equivalent concentrations. Inhibitionconstants (K_(i)) for MTX and its derivatives can be determined byplotting 1/V (v=velocity (μmole)) vs 1/ S! ( S!=substrate concentration,i.e., DHF) at different inhibitor concentrations, determining Km_(app)##EQU1## and using the equation ##EQU2## to solve for K_(i) ;(Km=Km_(app) at I!=O.

EXAMPLE V

Assay of the Inhibitory Activity of Methotrexate-DTPA on DihydrofolateReductase

The effects of modifying methotrexate with DPTA were unknown andrequired a comparison of the activity of equimolar concentrations ofmethotrexate and DTPA-MTX. Since the opterin ring had not been modifiedand no additional chromophores had been placed on methotrexate duringDTPA modification, the extinction coefficient of MTX (E=22,100 M⁻¹ cm⁻¹at 302 nm)) was used to determine the concentration of DTPA-MTX. Theinhibition of rhDHFR by MTX and DTPA-MTX were then measured under theassay conditions mentioned earlier and compared. FIG. 7 shows that at1×10⁻⁸ M and 5×10⁻⁹ M inhibitor concentration, the inhibitory effects ofDTPA-MTX were virtually identical to MTX inhibition; as indicated by thedecreased rates of dihydrofolate reduction.

EXAMPLE VI

Analysis of the Inhibitory Activity of MTX and DTPA-MTX on DHFR Bound inan Antibody DHFR Complex

As shown earlier, 16.88-DHFR conjugates have been prepared whichpossessed easily assayable quantities of rhDHFR. Although the reductaseactivity was measurable, there was no guarantee that the MTX bindingproperties of the conjugated enzyme had not been affected during themodification steps. To confirm that MTX binding was proportional to thedihydrofolate reductase activity in 16.88-DHFR, the DHFR activity in theconjugate was titrated to an equivalent amount of native rhDHFR andthese activity equivalents were compared for their ability to beinhibited by MTX and DTPA-MTX. FIG. 8 shows the results of MTXinhibition of equivalent activities of native rhDHFR and 16.88 boundrhDHFR and indicates that MTX binding is proportional to the reductaseactivities regardless of whether it is free or in conjugate form. Anidentical experiment performed using DTPA-MTX (FIG. 9) confirmed themethotrexate data. From these results, not only has the reductaseactivity been maintained in the conjugate, but also the ability of MTXand DTPA-MTX to bind to and inhibit the conjugated rhDHFR.

EXAMPLE VII

Synthesis of DTPA-MTX

Much effort has been devoted toward potent folate analogues and it iswell known that the glutamate moiety contributes to the binding of MTXto dihydrofolate reductase while the γ-carboxyl does not. We havedesigned MTX analogues that contain a chelator molecule at theγ-carboxyl group of the glutamic acid portion.

The synthesis of DTPA-MTX is shown schematically in FIG. 10. TheMTX-AB-GH was prepared using the general method of Rosowsky et al., J.Med. Chem., 1981, 24, 1450-1455. The DTPA dianhydride (9.3 mg, 25 μmol)was dissolved in DMF and stirred with Et₃ N (0.1 mL) for 5 min.MTX-AB-GH 6.8 mg (13 μmol) in 2 mL of CH₃ CN was added to the abovemixture and stirred overnight at room temperature. Solvents wereevaporated and the residue was heated to 50° C. with 1N HCl for 1 hour.The reaction mixture was evaporated to dryness and the residue waspurified by HPLC (a C₁₈, reversed-phase silica gel column, absorbance at280 nm, the mobile phase was formed with 2% acetic acid (pump A) and 2%acetic acid in 50% methanol (pump B); t_(R) =17.56 min (cf. t_(R) ofMTX=25.26 min) to give 4.1 mg (38%) of product; FAB-MS m/z=844 (M+H)⁺ ;¹ H NMR (D₂ O) δ 8.49 (s,1H), δ 7.52 (d,J=8.6 Hz, 2H) δ 6.72 (d, j=8.6Hz, 2H), δ 4.4 (m, 1H), δ 3.0-3.95 (m, 18H) δ 3.7 (s,2H), δ 3.1(s, 3H),δ 2.39(t, 2H), δ 1.9-2.3 (m, 2H).

EXAMPLE VIII

Clearance of ¹¹¹ In-DTPA-MTX from Athymic Mice Radiolabeling DTPA-MTXwith In-111

¹¹¹ InCl₃ (1.5 mCi) is mixed with 0.06 mL (0.6 mg) DTPA-MTX, 0.02 mL0.06 sodium citrate pH 5.5, and 0.01 mL 0.60 sodium acetate pH 5.5 for30 minutes to 215 minutes at room temperature. Thin layer chromatographyon plastic backed silica gel strips (1.1 ammonium acetate:methanol)using 0.001 mL of the final reaction solution showed greater than 95%incorporation of ¹¹¹ In into the ¹¹¹ In-DTPA-MTX complex. FIG. 11 showsthe migration of ¹¹¹ In-DTPA-MTX in the silica gel with an R_(f) of 0.5to 0.7. Free ¹¹¹ In does not migrate from the origin in this system.

This example compares the whole body clearance of ¹¹¹ In-DTPA-MTX withthat of ¹¹¹ In-DTPA. ¹¹¹ In-DTPA is known to clear rapidly from thecirculation with little retention in normal tissues. Clearance of the¹¹¹ In-DTPA-MTX at a rate similar to that of ¹¹¹ In-DTPA would indicatethat rapid body clearance of the portion of the conjugate not bound intumor tissue by antibody-DHFR may be expected. Such rapid clearancewould ensure that nearly all the unbound radionuclide would decayoutside the body.

Three athymic nu/nu mice were injected via the lateral tail vein with 50uCi ¹¹¹ In-DTPA-MTX in 0.5 mL 10% normal mouse serum in phosphatebuffered normal saline. A second group of three animals received 50 uCi¹¹¹ In-DTPA by the same route. All animal were examined for whole bodyretention of In-111 in a Capintec dose calibrator at 0.5, 1, 2, 3, 4,and 24 hours after injection.

Results shown in FIG. 12 indicate that the ¹¹¹ In-DTPA-MTX and the ¹¹¹In-DTPA clear from the mice at similar rates indicating the likelihoodof rapid urinary excretion of a DTPA-MTX not bound to antibody-DHFR.

EXAMPLE IX

Binding of¹¹¹ In-DTPA-MTX to Tumor Cell-Bound Antibody-DHFR

This example examines the targeting of ¹¹¹ In-DTPA-MTX to antibody-DHFRbound to K562 cultured erythroleukemia cells in vitro. For thisdemonstration, DHFR was coupled to the murine antibody to the humantransferrin receptor (5E9C11) using the methods described in example 1.This antibody rather than the human anti-colon carcinoma antibody 16.88was used since an antibody that binds to epitopes on the surface ofcultured cells was required for this in vitro demonstration.

In the first study binding of antibody-DHFR to the target cells wasassessed in a titration using concentrations of antibody-DHFR of 1.5,3.0, 6.0, 12.0, and 24.0 ug/mL mixed at 4° C. with 1×10⁶ K562 cells in amedium consisting of Hanks Balanced Salt Solution containing 1% bovineserum albumin (protease-free). The reaction volume was 0.2 mL reactedfor 60 minutes in an ice bath to prevent internalization of the antibodybound to the transferrin receptor. After washing away excess antibody,10 ng ¹¹¹ In-DTPA-MTX (0.53 uCi) was added and the reaction continuedfor 30 minutes in an ice bath.

Results of the titration are shown in FIG. 13. Non-specific binding ofthe ¹¹¹ In-DTPA-MTX, determined with antibody to which no DHFR wasconjugated, averaged 0.85% at all antibody concentrations. The studydemonstrated that ¹¹¹ In-DTPA-MTX can bind to antibody-DHFR bound totumor cells. The extent of binding was sufficient to saturate allavailable DHFR sites as determined from estimates of the amount of DHFRbound to the cells and the specific activity of the ¹¹¹ In-DTPA-MTX. Asno plateau level was reached it is apparent that at an antibody-DHFRconcentration of 24 ug/mL the available binding sites on the tumor cellswere not saturated in this study.

In the second study, the lowest concentration of antibody-DHFR givingsignificant binding above the background level (6.0 ug/mL) was used in atitration of the ¹¹¹ In-DTPA-MTX using concentrations of 12.5, 25, 50,100, or 200 ng/mL (0.31 to 3.7 uCi/mL) with conditions identical tothose described for the first study. Results are shown in FIG. 14. Againbinding of ¹¹¹ In-DTPA-MTX to cell-bound antibody-DHFR was demonstrated.At concentrations above 50 ng/mL, binding reached a plateau levelindicating saturation of the available DHFR binding sites, in agreementwith the conclusions of the first study.

EXAMPLE X

Stabilization of Dihydrofolate Reductase (rhDHFR)

In our most preferred embodiment we use stabilized rhDHFR (Dr. JamesFreisheim, Medical College of Ohio, Toledo, Ohio) as the enzyme. DHFRwas stabilized through covalent conjugation of the enzyme with aphotoaffinity analog of NADP⁺ (ANPAP-NADP) followed by photoactivationusing a tungsten halogen lamp (615 W; DVY; 3400° K).

A 10-fold molar excess of the photoaffinity analog was mixed with theenzyme, and the volume adjusted to a protein concentration of 1 mg/mlusing 10 mM Tris-HCl buffer (pH 7.5). The mixture was kept in ice at a10 cm distance from the light source while photoactivation was carriedout for 5 minutes with occasional stirring. An aliquot was assayed forDHFR activity before and after photoactivation. The NADP⁺ -linked enzymewas purified by gel-filtration using 10 mM sodium phosphate buffer, pH7.2, containing 20 mM NaCl. Fractions were assayed for DHFR activitybefore they were pooled for protein estimation (using Pierce BCAreagent) and determination of incorporation of NADP⁺ moieties per enzymemolecule.

Stability of the conjugated enzyme was determined by incubating at 37°C. for several hours. Percent stability was calculated by comparing witha sample kept at 4° C.

RESULTS

Conjugation

Photoactivation Time: 5 minutes

Percent remaining activity following photoactivation: 100%

Number moieties per enzyme molecule 2.21:1

    ______________________________________                                        Stability                                                                              Time (minutes)                                                       Sample     0       30      60   120   180  1080                               ______________________________________                                        ANPAP-NADP.sup.+ -                                                                       100.sup.1 %                                                                           N.T..sup.2                                                                            82.sup.1 %                                                                         78.sup.1 %                                                                          78.sup.1 %                                                                         67.sup.3 %                         rhDHFR                                                                        rhDHFR     100.sup.1 %                                                                           18.sup.1 %                                                                            10.sup.1 %                                                                         N.T.  N.T. N.T.                               ______________________________________                                         .sup.1 Values represent percent enzyme activity remaining after incubatio     at 37° C.                                                              .sup.2 N.T. -- Not Tested.                                                    .sup.3 Values represent percent enzyme activity remaining after incubatio     at 23° C.                                                         

Synthesis of the stabilizer of N3'-O-3-(4-azido-2-nitrophenyl)amino!propionyl NADP⁺ (ANPAP-NADP)

A modified procedure of the Chen and Guillory's method (Chen, S. andGuillory, R. J., J. Biol. Chem., 1980, 255, 2445-2453) was used and thedetailed procedure was as follows: A dimethyl formamide (DMF) solutionof carbodiimidazole (CDI) (324 mg, 2 mmol) and3-(4-azido-2-nitrophenylamino) propionic acid (Jeng, S. J. and Guillory,R. J., J. Supramolecular Structure, 1975, 3, 445-468) (15 mg, 0.6 mmol)was stirred at room temperature for 15 min. Then about 3 ml aqueoussolution of NADP⁺ (64.6 mg, 0.08 mmol) was added to the DMF solution.Stirring was continued overnight under a nitrogen atmosphere. Thesolvent was then removed by a rotary evaporator and the residue waswashed with acetone by centrifugation. The residue was then dissolved ina small amount of water and purified by preparative thin layerchromatography (Taper® plate; solvent system:1-butanol/water/HOAc=5/3/2). The material (R_(f) =0.4) was recovered.The compound was further purified by HPLC (Reverse Phase C₁₈ columnUV@260 nm). ##STR1##

We claim:
 1. A method for the in vivo targeting of an effector moleculein a patient comprising; first administering a targeting moiety coupledto an enzyme to form a targeting moiety-enzyme conjugate, wherein saidtargeting moiety has affinity for extracellular binding sites in atarget area, and thereafter administering a binding partner for theenzyme, wherein the binding partner is an enzyme inhibitor, coupled toan effector molecule forming an effector complex, whereby said effectorcomplex through the binding partner binds to the enzyme to localize saideffector molecule in the target area.
 2. The method of claim 1, whereinthe binding partner to the enzyme is an enzyme inhibitor.
 3. The methodof claim 1, wherein the enzyme is an intact enzyme, or a fragment orderivative of an enzyme that comprises the inhibitor binding region ofthe enzyme, or a molecule that mimics the inhibitor binding region of anenzyme, provided that the said enzyme will bind the inhibitor.
 4. Themethod of claim 1, wherein the targeting moiety is selected from thegroup consisting of an antibody, an antibody fragment, an antibodyvariable region, a complimentarity determining region of an antibody, abivalent antibody, a hybrid antibody and a chimeric antibody.
 5. Themethod of claim 1, wherein the targeting moiety is a ligand other thanan antibody that has receptors for the target area.
 6. The method ofclaim 1, wherein the effector molecule is a pharmacologically activecompound.
 7. The method of claim 1, wherein the effector molecule is aradionuclide or a toxin.
 8. The method of claim 7, wherein the effectormolecule is a radiometal.
 9. The method of claim 1, wherein the enzymeinhibitor is methotrexate and the effector molecule comprises aradiometal.
 10. The method of claim 1, wherein the effector complexcomprises methotrexate and a derivative ofdiethylenetriamine-pentaacetic acid according to the formula: ##STR2##wherein R¹ represents a group of the formula: ##STR3##
 11. The method ofclaim 1, wherein the enzyme is stabilized dihydrofolate reductase.